Detection method using kit for detecting plurality of target nucleic acids

Abstract

The present invention provides a method for detecting multiple target nucleic acids, the method making it possible to amplify a plurality of genes using one reaction vessel containing therein one type of reaction solution and further using a single label. Computation is performed in accordance with the following formulas for every specific cycle and / or every cycle during amplification reaction using a kit for detecting the multiple target nucleic acids. (Formula 1): f1[n]=fhyb.1[n] / fden.1[n], (Formula 1′): f2[n]=fhyb.2[n] / fden.2[n], (Formula 2): Fr[n]=(a−f2 [n]) / (a−f1 [n]).fhyb.1 [n]: Fluorescence intensity value in elongation step of first target nucleic acid detecting step. The same hereinafter.

Description

BACKGROUND OF THE INVENTION1. Field of the Invention[0001]The present invention relates to a detection method using a kit for detecting multiple target nucleic acids, the kit improving the PCR method and further measuring the multiple target nucleic acids.2. Description of the Related Art[0002]In genetic screening, it is necessary to measure multiple target nucleic acids in many cases. For example, the multiple target nucleic acids may be: a first pair of influenza type A and influenza type B; a second pair of influenza and RSV / human metapneumovirus; a third pair of Chlamydia and Nisseria gonorrhoeae, which may cause sexually transmitted diseases; a fourth pair of Mycoplasma and resistance factor thereof; and so on.[0003]Regarding the multiple target nucleic acids in some pairs of the above, the conditions of patients are similar to each other and also infection of the same expands simultaneously. So, it is conceivable to distinguish the multiple target nucleic acids from each other...

AI Technical Summary

Benefits of technology

The present invention provides a kit and a detection method for multiple target nucleic acids using a single reaction vessel and a single label. The kit includes a solution containing a first and second target nucleic acid, a first and second target primer, a first and second target probe, DNA polymerase, deoxyribonucleoside triphosphate, and a second target probe. The detection method involves calculating the fluorescence intensity of the first and second targets in real-time during amplification and denaturation steps. The kit and method provide a reliable and efficient way to detect multiple target nucleic acids simultaneously.

Problems solved by technology

The first process of performing the independent measurement requires long time and high costs.

The second process of distinguishing different labeling substances from each other costs too much because the second process needs not only preparing plural kinds of labeled reagents but also complicated wavelength-setting in an analyzer.

There is a problem that the method requires a long time.

In other words, the analyzer costs too much.

This is a serious problem.

If the temperature is changed speedily upon the melting curve analysis, it becomes difficult to identify melting temperature for each gene.

Accordingly, there is a problem that extra time of about 5 to 10 minutes after PCR is required.

Accordingly, too many conditions should be taken into consideration, design of the profile is also difficult, and time for measurement must be too long.

Furthermore, there is another problem that temperature must be drastically changed to task the device.

As mentioned above, a common problem upon simultaneously amplifying multiple genes by means of one reaction vessel containing one kind of reaction solution is that fluorescent signals interfere with each other to make distinction there-between impossible caused by base sequences of target genes and / or probes.

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